Project Summary Cancer cells have a distinct metabolism that supports their pathological proliferation. Targeting this metabolism has led to many of the existing chemotherapies in the clinic today such as antifolates. The amino acid glycine is primarily produced by folate metabolism, a pathway that is nearly universally upregulated in cancer. Glycine supports important biosynthetic processes including protein, purine and glutathione synthesis. In addition, glycine catabolic processes have recently been shown to be important in cancer stem cells and glioblastoma. In newly published data, I demonstrated that glycine-supported antioxidant production is crucial for cancer cell growth. This proposal seeks to quantitatively characterize glycine metabolism in cancer and to test the hypothesis that folate-mediated glycine synthesis supports glutathione production and thus redox homeostasis. In these efforts, I will benefit from a new small molecule that I discovered, which potently inhibits both isozymes of the glycine synthetic enzyme SHMT. In Aim 1, I will use isotope tracers to quantitate glycine production and consumption fluxes in normal and cancer cells, and apply these findings in vivo. In Aim 2, I will further develop the SHMT inhibitor and test it in a mouse model of B-cell lymphoma, a tumor type that is defective in glycine import and accordingly highly sensitive SHMT inhibition in vitro. Collectively, these experiments will lay the groundwork for targeting of glycine metabolism as a new therapeutic approach in cancer. Under the guidance of my primary mentor and my team of collaborators during the K99 period, this proposal will allow me to successfully transition from my current mentored position into a independent research group leader.